Biochimica et Biophysica Acta (BBA) - General Subjects
Membrane-associated protein kinases in phorbol ester-activated human polymorphonuclear leukocytes
Abstract
Membrane-associated protein kinases in human polymorphonuclear leukocytes were studied. In unstimulated polymorphonuclear leukocytes the protein kinase C was predominantly present in the cytosol but in phorbol 12-myristate 13-acetate-(PMA-) activated cells a time and dose-dependent translocation of the kinase to the particulate fraction occurred. Two new protein kinase activities also appeared in the particulate fraction upon PMA activation. The one had a Mr of 40 000 and its activity was independent of phospholipids. The other (Mr 90 000) was partially activated by phospholipids, but separated from protein kinase C on DEAE-cellulose chromatography.
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Cited by (15)
Dissociation of mitogen-activated protein kinase activation from the oxidative burst in differentiated HL-60 cells and human neutrophils
1995, Journal of Biological ChemistryIn human polymorphonuclear leukocytes (PMNs), mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (Erks), are activated within minutes upon stimulation with either chemoattractant formyl-Met-Leu-Phe (fMLP) or phorbol 12-myristate 13-acetate (PMA). This activation of MAPKs coincides with the formation of superoxide anion, which occurs through the activation of a multiple-component NADPH oxidase pathway. MAPKs have thus been suggested to be involved in signal transduction leading to the oxidative burst. To investigate whether MAPK activation plays a central role in the oxidative burst, we evaluated the effect of cAMP on MAPK activation induced by fMLP and PMA. cAMP inhibits many PMN functional responses, including the oxidative burst, and has recently been shown to reduce growth factor- and PMA-induced MAPK activities in a variety of cells. We found that in differentiated, neutrophil-like HL-60 cells, while cAMP reduced PMA-induced MAPK activation, it had no effect on fMLP-induced MAPK activation. Despite the presence of unchanged levels of activated MAPKs, the fMLP-induced oxidative burst was substantially diminished by cAMP. By contrast, O2- production induced by PMA remained the same even though MAPK activation was inhibited. In PMNs, although the levels of O2- induced by either 10 ng/ml or 100 ng/ml PMA were similar, only 100 ng/ml could stimulate MAPK activation, suggesting that the oxidative burst could occur in the absence of detectable activation of MAPKs. As in HL-60 cells, cAMP inhibited the O2- production in fMLP-stimulated PMNs but had no effect on MAPK activity. These results demonstrate that, while MAPK activation coincides with PMN activation, it can be dissociated from the oxidative burst.
Effects of tripeptides derived from milk proteins on polymorphonuclear oxidative and phosphoinositide metabolisms
1992, Biochemical PharmacologyThe tripeptide GLF (glycyl-leucyl-phenylalanine) was isolated from human milk proteins. This peptide increased phagocytosis by human and murine macrophages and protected mice against Klebsiella pneumonia infection. Specific binding sites on human polymorphonuclear leukocytes (PMNs) have been demonstrated recently. The aim of the present research was to study the action of this peptide on rat and human PMN oxidative burst and to investigate the consequences of cell stimulation on polyphosphoinositide hydrolysis. A biphasic stimulating concentration-dependent effect of GLF on PMN chemiluminescence and Superoxide anion generation was demonstrated. One of the peaks of the oxidative response occurred around 10−9 M, which correlates with the Kd of high affinity receptors of GLF. The other maximum, around 10−4 M, might be due to the hydrophobic nature of the tripeptide. O−2 generation mimicked the phorbol myristate acetate response: after a lag period of 2–5 min. O−2 release gradually increased for 10–15 min until a plateau was reached. Furthermore, GLF enhanced phosphoinositide breakdown with maximal IP3 production at 10−7 M. Various analogs of GLF were synthesized in order to define the relative importance of the different amino acids and their position in the tripeptide molecule: glycyl-phenylalanine-leucine was devoid of biological properties but enhanced the activity of GLF on the metabolic burst at high concentrations; peptides leucyl-leucyl-phenylalanine and leucyl-leucyl-tyrosine, which displaced GLF from its specific membrane receptors, exerted stimulating effects on PMN oxidative and phosphoinositide metabolisms. It is quite conceivable that these short peptides, which may be generated in the newborn during digestion and which are able to stimulate phagocytic cells, are implicated in the defense of the neonate immature organism against infection.
Heat shock induces alterations of the lipoxygenase pathway in human polymorphonuclear granulocytes
1989, Prostaglandins, Leukotrienes and Essential Fatty AcidsWe have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42°C. Subsequently, the different test systems were performed at 37°C. As we confirmed by the release of lactate dehydrogenase and β-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca++-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42°C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.
A time-course study on superoxide generation and protein kinase C activation in human neutrophils
1988, FEBS LettersThe time course of superoxide generation and membrane association of protein kinase C was studied in human neutrophils stimulated by PMA, FMLP, ionomycin and A23187. The initiation of superoxide generation in PMA; ionomycin- and A23187-stimulated neutrophils was characterized by a lag period of at least 30 s in contrast to a lag period of 10–15 s in FMLP-stimulated cells. The time course of membrane association of protein kinase C in PMA-stimulated neutrophils was highly dependent upon the PMA concentration used for stimulation. However, membrane association of protein kinase C preceded superoxide generation in cells stimulated by 10–300 ng/ml PMA. FMLP, ionomycin and A23187 induced membrane association of protein kinase C in a few seconds and always before superoxide generation. It is concluded that membrane association of protein kinase C in PMA-, FMLP-, ionomycin- and A23187-stimulated neutrophils precedes superoxide generation, and thereby may be part of the mechanism initiating NADPH-oxidase activity. A simple correlation between the two parameters could not be proven, indicating that also other activation mechanisms are decisive in the activation of NADPH-oxidase.
A study on the role of protein kinase C and intracellular calcium in the activation of superoxide generation
1988, BBA - Molecular Cell ResearchAccumulating evidence indicates that protein kinase C plays an essential role in activation of NADPH oxidase. In the present study, the correlation between superoxide generation, intracellular calcium, activation of purified protein kinase C and stabilized membrane-bound protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) and 1-deacyl-2-acetyl-rac-glycerol (OAG) were found to induce equal activation of purified protein kinase C and translocation of protein kinase C to the membrane fraction, but differed significantly in their ability to induce superoxide generation. Intracellular calcium was varied using calcium ionophores and increasing the intracellular calcium concentration to more than 1 μM was found to induce increased superoxide generation in maximally OAG-stimulated cells; this contrasted to maximally PMA-stimulated leukocytes. Ionomycin and A23187 were both found to induce a translocation of protein kinase C to the membrane fraction. This translocation was highly dependent upon extracellular calcium. In contrast, PMA- and OAG-induced translocation of protein kinase C was not dependent upon extracellular calcium. In conclusion, our results indicate that although PMA, OAG and calcium ionophores seem to activate protein kinase C in human polymorphonuclear leukocytes these activators differ in their ability to induce superoxide generation.
Protein kinase C activity in activated human T-lymphocytes stimulated by interleukin-2
1988, BBA - Molecular Cell ResearchInterleukin-2 and phorbol 12-myristate 13-acetate (PMA) are shown to induce DNA-synthesis in human T-lymphocytes activated with phytohaemagglutinin. However, whereas PMA induced a rapid and persistent translocation of protein kinase C from cytosol to particulate fraction, no translocation was observed upon stimulation with interleukin-2. Treatment with PMA for 72 h caused a slow down-regulation of protein kinase C activity to less than 10% of unstimulated T-lymphocytes and was mainly located in the particulate fraction. In contrast, stimulation with phytohaemagglutinin increased the total cellular protein kinase C activity by approx. 100% but with an unaltered subcellular distribution. However, interleukin-2-induced DNA synthesis in PMA- and phytohaemagglutinin-stimulated T-lymphocytes was comparable. Further, maximal DNA synthesis was shown to be dependent on the continuous presence of interleukin-2. These results indicate that interleukin-2-induced proliferation of activated human T-lymphocytes can occur without a translocation of protein kinase C from the cytosol to the particulate fraction and that interleukin-2 most likely functions as a progression factor.