Protein phosphorylation in intact coronary endothelial cells by bradykinin

In cultured coronary endothelial cells obtained from guinea pig hearts, bradykinin (10(-6) M) stimulated the 32Pi-incorporation into 5 substrate proteins with molecular weights corresponding to 27, 32, 60, 86 and 100 kDa. The time course of phosphorylation of the 60, 86 and 100 kDa proteins was rapid (within 30 s), but transient (max. within 1-2 min.), while the 32Pi incorporation into the 27 and 32 kDa protein was delayed but increased within 10 minutes. Ca+(+)-ionophore A 23187 (10(-5) M) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-5) M) both mimicked the effects of the bradykinin induced phosphorylation pattern. While A 23187 enhanced the phosphorylation of the 27, 60 and 100 kDa substrates, TPA increased the 32Pi-incorporation into the 32 and 86 kDa proteins. Furthermore the time course of protein phosphorylation elicited by A 23187 and TPA showed marked similarities to those obtained with bradykinin. Our findings are consistent with the view, that stimulation of coronary endothelial bradykinin-receptors activates both Ca+(+)-dependent protein kinases and protein kinase C.     

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils.

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Stimulation of the neutrophils with chemotactic factor fMet-Leu-Phe (10 nM) caused rapid increases of tyrosine phosphorylation of several proteins with apparent molecular masses of (Group A) 54-58 kDa and 100-125 kDa and (Group B) 36-41 kDa. Stimulation of Group A proteins was observed by fMet-Leu-Phe (10 nM, maximum at 20 s) and A23187 (1 microM, 1 min). Stimulation of Group B proteins was observed by fMet-Leu-Phe (ED50 0.15 nM, 1 min), leukotriene B4 (ED50 0.15 nM, 1 min), phorbol 12-myristate 13-acetate (PMA) (ED50 25 ng/ml, 10 min) and partially by ionophore A23187 (1 microM, 1 min). Pretreatment of the cell with the protein kinase inhibitor H-7 (25 microM, 5 min) and PMA (0.1 microgram/ml, 3 min) partially inhibited the fMet-Leu-Phe effect. However, pretreatment of the cells with quin 2/AM (20 microM, 10 min) completely inhibited the fMet-Leu-Phe effect. The results indicate that rapid regulation of tyrosine phosphorylation is an early event occurring in stimulated neutrophils. Furthermore the effect of fMet-Leu-Phe on tyrosine phosphorylation may require Ca2+ mobilization and may partially require the activity of H-7-sensitive protein kinases.     

Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor

The effects of short-term phorbol ester treatment of CHO cells that stably express 900 fmol of recombinant human serotonin 5-HT1A receptor/mg of protein on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the receptor were studied. Pretreatment of cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a dose- and time-dependent shift of the half-maximal dose of serotonin (5-HT) required to inhibit membrane adenylyl cyclase (from IC50 approximately 100 nM to approximately 400 nM). This desensitization (shift in IC50) was rapid, occurring with 5 min of pretreatment and being maximal by 10-15 min; it was also dose-dependent, being half-maximal at approximately 300 nM PMA. Desensitization was also induced by sn-dioctanoylglycerol (DiC8) and blocked by the protein kinase C (PKC) inhibitors sphingosine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). In detached permeabilized cells, PMA pretreatment caused a rapid phosphorylation of immunoprecipitated 5-HT1A receptors, with an approximately 3-4-fold increase that was maximal after 15 min and persisted for 90 min. The phosphorylation occurred at a similar dose of PMA as that which induced desensitization (half-maximal at approximately 300 nM, maximal at 500 nM to 1 microM), could be reproduced by pretreatment with the PKC activators DiC8 or phorbol 12,13-dibutyrate (PDBu), and could be blocked by the PKC inhibitors sphingosine or H-7. The stoichiometry of the phosphorylation was approximately 2 mol of [32P]ATP/mol of receptor, suggesting the involvement at least two of three putative PKC sites within the 5-HT1A receptor. The close concordance between the PKC-induced desensitization and phosphorylation suggests a potential causative link between these two effects of PKC on the human 5-HT1A receptor.     

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.     

Phosphorylation and dephosphorylation of soluble proteins in human eosinophils

The effect of phorbol 12-myristate 13-acetate (PMA), calcium ionophore (A23187), opsonized zymosan (OZ), and N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) on protein phosphorylation was examined in purified eosinophils (eos) isolated from human peripheral blood. Eos were prelabeled with [32P]orthophosphate, stimulated with several activating agents for varying periods of time. The soluble proteins were then analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. In resting eos, there was phosphorylation of endogenous soluble proteins with molecular weights of 12, 16, 21, 40, and 66 kilodaltons (kDa). PMA, a potent activator of oxidative metabolism, induced phosphorylation of 19-, 40-, and 67-kDa proteins. A23187, a strong degranulating stimulus, caused phosphorylation of 40-, 53-, and 67-kDa proteins. OZ, a relatively weak stimulus for eos function, caused phosphorylation of 30-34-, 59-, 67-, and 93-kDa proteins. In addition, all the above stimuli caused a time-dependent dephosphorylation of 21-kDa protein. In contrast, f-Met-Leu-Phe caused neither phosphorylation of new proteins nor dephosphorylation of preexisting eos proteins. These findings demonstrate that selected stimuli affect phosphorylation of soluble eos protein. These results also suggest that phosphorylation of specific proteins in eos is an intermediary step in external stimulus-induced cell activation, which may involve many different cell functions.     

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.     

Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells

ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).     

Calcium transport and phosphorylated intermediate of (Ca2+ + Mg2+)-ATPase in plasma membranes of rat liver

We have identified and characterized calcium transport and the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in plasma membrane vesicles prepared from rat liver. The calcium transport did not absolutely require the presence of oxalate and was completely inhibited by 1 microM of ionophore A23187. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle and liver microsomes, was not absolutely required to maintain the net accumulation of calcium. The Vmax and Km for calcium uptake were 35.2 +/- 10.1 pmol of calcium/mg of protein/min, and 17.6 +/- 2.5 nM of free calcium, respectively. Ten mM magnesium was required for the maximal accumulation of calcium. Substitution of 5 and 10 mM ADP, CTP, GTP, and UTP for ATP could not support calcium uptake. The calcium uptake was not affected by 0.5 mM ouabain, 20 mM azide, or 2 micrograms/ml of oligomycin but was inhibited in a dose-dependent fashion by vanadate, with a Ki of approximately 20 microM for vanadate. The substrate affinities and specificities of this calcium-transport activity suggest that it is closely associated with the (Ca2+ + Mg2+)-ATPase reported in the plasma membranes of liver (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). A calcium-stimulated and magnesium-dependent phosphoprotein was also demonstrated in the same membrane vesicles. The free calcium concentration at which its phosphorylation was half-maximal was 15.5 +/- 5.6 nM. Sodium fluoride, ouabain, sodium azide, oligomycin, adriamycin, and N,N'-dicyclohexylcarbodiimide did not affect its formation while vanadate at 100 microM inhibited the calcium-dependent phosphorylation by approximately 60%. The properties of this phosphoprotein suggest that it may be the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in the plasma membranes of rat liver.     

Enhanced phosphorylation of progesterone receptor by protein kinase C in human breast cancer cells

Affinity-isolated progesterone receptor (PR) from human breast cancer cells incubated with [32P]orthophosphate was shown to exist as a phosphoprotein. Exposure of the cells to 10 nM phorbol-12-myristate-13-acetate (PMA) for 10 min increased by 30-40% the amount of label incorporated into the 116-kDa receptor protein. A two-fold increase in the total number of steroid binding sites was also observed in cells receiving PMA treatment. This apparent unmasking of PR binding sites by phosphorylation probably involved conformational changes to existing receptor complexes and affected the eventual state of receptor dissociation or transformation. An increase primarily in the 8 S sedimenting molecular species was observed but PMA treatment also led to the appearance of a smaller, 2-3 S form of receptor (10% of total) that was not present in control samples. When cytosols were partially transformed in vitro by ATP and salt, all molecular species of receptor (8, 4, and 2-3 S) from the PMA-treated samples consistently migrated faster in sucrose gradients. The larger amount of 2-3 S receptor in PMA-treated samples disappeared when ATP, but not salt, was the transforming agent. These results suggest a major role for phosphorylating reactions in the receptor-mediated action of steroids by regulating hormone-binding and influencing receptor transformation. Tumor promoters such as the phorbol esters may act by artificially increasing the level of processing of steroid receptor.     

Serine protein kinase activity associated with rotavirus phosphoprotein NSP5.

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [gamma-32P]ATP resulted in 32P labeling of 28- to 35-kDa NSP5. The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.     

An epsilonPKC-selective inhibitor attenuates back phosphorylation of a low molecular weight protein in cardiac myocytes

We have studied epsilon PKC-mediated phosphorylation events in neonatal cardiac myocytes using back phosphorylation. 3 nM 4-beta 12-myristate-13-acetate (PMA)-intact cell treatment preferentially activates epsilon PKC in these cells (Circ. Res. 76 (1995) 654) and caused decreased 32P incorporation (back phosphorylation) into an approximately 18-kDa protein. This response required physiological levels of free Mg(2+) and short (3-5 min) incubation periods in back phosphorylation assays. Introduction of a selective epsilon PKC translocation inhibitor (epsilon V1) into these cells attenuated the 3 nM PMA-induced back phosphorylation response while translocation inhibitors to the classical PKC or deltaPKC isozymes were without effect. Pretreatment of our cells with endothelin-1 (ET1) had similar effects to 3 nM PMA albeit the magnitude of the ET1 back phosphorylation response was about one-half that of 3 nM PMA. Our results suggest that epsilon PKC phosphorylates an approximately 18-kDa protein found in the particulate cell fraction of neonatal cardiac myocytes. Epsilon PKC modulates diverse cardiac responses including contraction, ion channel functions, hypertrophy, and ischemic preconditioning. Characterization of epsilon PKC-selective phosphotransferase events may reveal novel regulatory mechanisms for this enzyme in neonatal cardiac myocytes.     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: Further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72-kDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with ³²P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514–523. © 1997 Wiley-Liss, Inc.     

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.     

Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils.

Phosphorylation of a 47 kDa protein in human neutrophils is induced by phorbol 12-myristate 13-acetate (PMA), opsonized latex beads, fMet-Leu-Phe, calcium ionophore A23187 and fluoride. All of these stimuli activate the specialized microbicidal respiratory burst of neutrophils, and in each case the kinetics of activation correspond with the kinetics of phosphorylation of the 47 kDa protein. Trifluoperazine (50 microM) and chlorpromazine (100 microM), inhibitors of calmodulin and protein kinase C, abolish the increase in oxygen consumption and selectively prevent phosphorylation of the 47 kDa protein after PMA stimulation. Treatment of neutrophils with pertussis toxin totally inhibits both superoxide production and phosphorylation of this protein in response to fMet-Leu-Phe, but not in response to PMA, indicating that a GTP-binding protein modulates the fMet-Leu-Phe receptor signal. Phosphorylation of the 47 kDa protein, a phenomenon absent from the neutrophils of subjects with autosomal recessive chronic granulomatous disease, which lack the respiratory burst, appears to be the common trigger for activation of the burst in normal neutrophils.     

Extracellular adenosine nucleotides stimulate protein kinase C activity and human neutrophil activation

We studied the effect of adenosine nucleotides on several aspects of the functional activation of human peripheral blood polymorphonuclear leukocytes (PMN). Radiolabeled ATP bound to PMN in a manner suggesting the existence of specific binding sites because: 1) binding was reversed (92 +/- 6%) by 100-fold excess concentrations of unlabeled ATP but minimally by either ADP (43 +/- 12%) or GTP (37 +/- 8%); and 2) binding saturation was achieved (i.e., specific binding did not increase) above 250 microM ATP. Binding studies revealed that significant ATP hydrolysis occurred, even at low temperatures and in the presence of phosphatase inhibitors. Adenosine nucleotides activated signal transduction mechanisms in PMN because: 1) 1 to 100 microM ATP and 5'-adenylylimidodiphosphate (AMP-PNP) stimulated increased production of 1,2-diacylglycerols; 2) ATP (0.5 to 500 microM) and ADP (0.1 to 10 mM) induced increased insoluble protein kinase (PKC) activity in a dose-dependent manner when used at concentrations greater than 50 microM; 3) ATP (greater than or equal to 50 microM) induced a shift in the solubility of phorbol receptors from mostly soluble (89% in untreated cells) to mostly insoluble (68%), whereas ADP, GTP, and GDP were effective at higher concentrations; and 4) greater than or equal to 50 microM ATP stimulated increased phosphorylation of endogenous PMN proteins. AMP-PNP induced PKC activity and phosphoprotein changes that were qualitatively similar to those observed when PMN were treated with ATP, suggesting that extracellular ATP hydrolysis is not required for signal transduction to activate PKC. Functionally, ATP stimulated the secretion of specific (but not azurophil) granules because vitamin B12-binding protein and low levels of lysozyme, but not beta-glucuronidase, were released; qualitatively similar results were obtained by using AMP-PNP. These results suggest that certain adenosine nucleotides employed at physiologically relevant concentrations stimulate increased 1,2-diacylglycerol production, PKC activity, granule secretion, and endogenous phosphoprotein formation in a manner that is independent of extracellular ATP hydrolysis.     

1,2-dioctanoyl-sn-glycerol can stimulate neutrophils by different mechanisms. Evidence for a pathway that does not involve phosphorylation of the 47-kDa protein

Neutrophils treated with 1,2-dioctanoyl-sn-glycerol (DiC8) are known to release large quantities of superoxide (O2-) and to exhibit an intense phosphorylation of two proteins with molecular masses of approximately 47 and 49 kDa. In this paper, we report that O2- release from guinea pig cells stimulated with a near optimal amount of DiC8 (2.0 microM) is markedly inhibited (greater than or equal to 70%) by antagonists of protein kinase C (i.e. 150 nM staurosporine; 200 microM 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7], whereas that from cells stimulated with an optimal amount of DiC8 (7.8 microM) is not (approximately 25% inhibition). However, staurosporine (150 nM) effectively reduced the level of phosphorylation of the 47- and the 49-kDa proteins to that observed in unstimulated cells when either amount of DiC8 (i.e. 2.0 or 7.8 microM) was utilized. Thus, neutrophils stimulated with 7.8 microM DiC8 in the presence of staurosporine release large quantities of O2- without an enhanced phosphorylation of the 47- and the 49-kDa proteins. In contrast, these antagonists of protein kinase C effectively blocked O2- release from neutrophils stimulated with an optimal amount of phorbol 12-myristate 13-acetate (PMA), and the percentage of inhibition was not affected by increasing the concentration of PMA 160-fold. These data show that DiC8 and PMA, both activators of protein kinase C, can have distinct effects on O2- release by neutrophils. Moreover, they suggest that DiC8 (or a metabolite) under certain circumstances may function in a stimulatory pathway for O2- release that is independent of protein kinase C. Differences in the morphology of neutrophils stimulated with PMA and DiC8 are presented. Ancillary data on human neutrophils are also provided.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Blue-light-induced phosphorylation of a plasma-membrane protein from phototropically sensitive tips of maize coleoptiles

Tips of maize coleoptiles, which function as esential light sensors for the phototropic growth reaction, exhibit a rapid blue-light-induced phosphorylation of a plasma-membrane-associated 100-kDa protein. Characteristics of this reaction are as follows: (i) The functional unit involved in the light-dependent phosphorylation consists of a photoreceptor, a protein kinase and the 100-kDa protein. This complex is only localized in the plasma membrane of tips but not in other parts of the seedling, (ii) The photoreceptor is a cryptochrome-like compound, (iii) The pH optimum of the light-dependent phosphorylation on isolated plasma membranes is around pH 7.8 whereas the light-independent phosphorylation of other membrane proteins occurs at lower values (pH 6.2). (iv) The light-induced in-vitro phosphorylation of the 100-kDa protein is strongly inhibited by the protein-kinase inhibitor staurosporine (IC₅₀=4 nM). (v) The ³²P-moiety of a ³²P-[100 kDa]-protein complex generated after a light pulse with the aid of a membrane-associated protein kinase in the presence of [γ-³²P]ATP cannot be removed by a 100-fold higher level of (unlabelled) ATP. This fact indicates that protein and phosphate are covalently connected and that the complex is not a short-lived intermediate. (vi) The 100-kDa protein is not identical to the plasma-membrane H⁺-ATPase, as shown by immunostaining on Western blots. (vii) Irradiation-dependent in vivo phosphorylation of the 100-kDa protein in tips is already saturated by a light pulse of 5 s. In contrast, the de-phosphorylation of the protein in the dark is a slow reaction lasting about 30 min. It is suggested that the blue-light-triggered phosphorylated status of the 100-kDa protein is an early step in phototropism of the coleoptile, affecting the transport of auxin primarily in the irradiated flank.